Migraine is one of the most common neurological disorders with over 29 million Americans suffering from the debilitating headaches and sensory disturbances that accompany a migraine attack. There is no cure for migraine and current treatment options are often ineffective or leave patients suffering from significant adverse side effects. While the underlying cause of migraine remains unknown many patients report an aura that is often perceived as a visual disturbance of flashing lights or blind spots. A neurological phenomenon known as cortical spreading depression (CSD) may cause migraine aura leading to the development of migraine pain. Migraine patients often identify specific migraine triggers including stress, alcohol, diet, the menstrual cycle and pregnancy. These triggers are also known to increase levels of neurosteroids in the brain. Neurosteroids can be synthesized directly in the brain or from peripherally produced sex steroids and can influence neuronal excitability through modulation of inhibitory GABAergic function. Increased neuronal excitability has been reported in migraine patients and can lower the threshold for developing CSD. While the effects of sex steroids in migraine have been extensively studied there is a critical gap in th understanding of how brain-derived neurosteroids may directly influence migraine. This proposal focuses on the actions and mechanism of neurosteroid induced changes to excitability and how they may impact CSD and migraine. We hypothesize that the effects of neurosteroids are cell type dependent and by selectively enhancing cortical inhibition paradoxically decrease the threshold for the development of CSD. Three specific aims test this hypothesis and the underlying mechanism utilizing cutting-edge advanced electrophysiological, imaging, optogenetic and LSPS circuit mapping techniques in an established animal model of CSD. First, how do neurosteroids differentially affect excitatory and inhibitory neurons? (Aim 1); second, what is the mechanism of neurosteroid mediated enhancement of CSD? (Aim 2); and third, do neurosteroids disinhibit the cortex and amplify synaptic circuits? (Aim 3). Our preliminary data demonstrate that neurosteroids decrease the threshold for CSD potentially through a select action on fast-spiking interneurons that reduces inhibitory drive onto excitatory pyramidal neurons. The data from the proposed experiments will provide insights into the pathophysiology of migraine as well as a mechanism through which neurosteroids may be a common mediator for several migraine triggers. Additionally, we will examine if specific neuronal populations can be targeted to prevent or inhibit CSD. These findings will allow us to identify mechanisms that may lead to enhanced excitability in migraine patients and potentially novel targets for therapeutic intervention to prevent or minimize migraine headache.